INTRODUCTIONRegulatory agencies have clearly established the need to identify and control harmful chemicals that may leach from container closure systems into the final packaged drug product. SGS Life Science Services performs extract-ables studies to identify those potential leachables in containers used in packaging and processing. A leachables study is necessary when extractables are present at concentrations above established safety limits. Analytical methods are then developed and validated to determine the presence of potential leachables in the drug product within the associated container. Once methodology has been established, the drug product and packaging mate-rial/container is placed on stability and monitored under pre-determined conditions for a set period of time.

One area of interest with respect to extractables and leachables is Biotherapeutics. Biotherapeutic products typi-cally consist of a bio-molecule formulated in a buffering solution with excipient(s). Polymeric single-use biopro-cess containers are becoming more common in the manufacturing of Biotherapeutic products. Extractable profiles of these polymeric materials are necessary to support the safety of their use in various therapeutic applications. Method development for the targeted extractables in Biotherapeutic formulations presents a number of analytical and technical challenges.

Tween (Polysorbate) 80 is a common surfactant used to prevent protein denaturation and aggregation in large mol-ecule drug formulations. The presence of Tween 80 makes analysis of small molecule contaminants very difficult. Liquid-liquid extractions are not feasible as the Tween emulsifies the hydrophobic and hydrophilic layers, rendering efficient phase separation impossible.

1. OBJECTIVESThis poster demonstrates the impact of Tween 80 on different separation and detection techniques.

2. MATERIALS & METHODSTween (Polysorbate)

Polysorbates are a category of excipients that are commonly used in Biopharmaceutical Formulations. They are nonionic surfactants composed of fatty acid esters of polyoxyethylene sorbitan. Tween is one brand name of polysorbate.

TWEEN 80 (POLYSORBATE 80)

ADVANTAGES:

• Readily soluble in aqueous matrices• Prevent biomolecules from aggregation• Minimizes surface adsorption to polymeric bioprocess containers

DISADVANTAGES:

• Degrades through auto-oxidation• Interferes with analytical separation methodologies in determining targeted analyte in biological formulations (solvent-solvent extraction and solid-

phase extraction)

BIOPROCESS MATERIAL EXTRACTION: VOLATILE ORGANIC COMPOUNDS

• Approximately 4 g of film material was cut into small pieces, placed into a headspace vial and sealed with a Teflon® coated crimp cap. • Sample preparations were analyzed by headspace gas chromatography with mass spectrometry (MS) and flame ionization detection, as indicated

(Headspace-gas chromatography-mass spectrometry [HS-GC-MS] or headspace-gas chromatography-flame ionization detection [HS-GC-FID]).

BIOPROCESS MATERIAL EXTRACTION: SEMI-VOLATILE AND NON-VOLATILE ORGANIC COMPOUNDS

• Approximately 15 g of minced film material was placed into a 250-mL round-bottom flask. Aqueous ethanol (150 mL; 20% v/v) was added to the flask. The system was equipped with a heating element and a reflux condenser. The system was allowed to reflux (temperature approximately 70°C) for 24 hours.

• Ethanol was removed from the extract solutions by distillation. The remaining aqueous phase was extracted with n-hexane. The organic (hexane) phases were withdrawn from the aqueous phase and dried with a small quantity of anhydrous sodium sulfate (Na2SO4).

• Semi-volatile organic compounds in the extracts were analyzed by gas chromatography with mass spectrometry and flame ionization detection (GC-MS and GC-FID, respectively).

• Semi-volatile and non-volatile organic compounds were analyzed by high performance liquid chromatography with ultra-violet and mass spectrom-etry detection (LC-UV-MS).

BIOPROCESS MATERIAL EXTRACTION: SOXHLET WITH N-HEXANE

• Approximately 15 g of minced film material was placed into a Soxhlet thimble. The Soxhlet apparatus was set up with 200 mL of n-hexane and al-lowed to extract for 24 hours.

• The hexane extract was then evaporated to a volume of 30 mL and analytically transferred to a final volume of 50 mL with n-hexane. • For semi-volatile organic compounds, turbidity was observed in the sample extract and centrifuged prior to analysis using GC-MS and GC-FID. • For semi-volatile and non-volatile organic compounds, high performance liquid chromatography with ultra-violet and mass spectrometry detection

(LC-UV-MS) was used.

POTENTIAL LEACHABLES: ANALYTICAL THRESHOLD TARGETED AT 150 PPB

• Irgafos 168 • 2,4-Di-tertbutyl Phenol• 1,3-Dt-Tertbutyl Benzene

3. RESULTS

4. CONCLUSION• GC-MS: Tween is not compatible with the GC system and cannot achieve desirable detection levels.• GC-HS: The targeted analyte could not be volatilized at 0.15 ppm (5 mL with 1 mL injection).• Purge and Trap-GC: Target analyte volatilization was not feasible because of Tween 80-related foaming.• Analytical separation methodologies, such as liquid-liquid extraction and solid-phase extraction, could not overcome problems introduced by Tween

80.• Further analytical development is necessary to determine extractables in a sample matrix with Tween 80. • In the future, we will perform base hydrolysis of Tween 80 yielding oleic acid as a byproduct. The hydrolysis of Tween 80 is a potential solution to

this problem. However, a potential complication is that the strong base may also degrade the analytes*.

REFERENCE*J. of Chrom B. “A simple reversed phase high-performance liquid chromatography method for polysorbate 80 quantitation in monoclonal antibody drug products.” Michael Adamoc, Lawrence W. Dick Jr. a, Difei Qiua, An-Horng Leea,John Devincentisb, Kuang-Chuan Chenga.

W5302 ANALYTICAL APPROACH TO DETECTING PACKAGING LEACHABLES IN BIOPHARMACEUTICAL PRODUCTS FORMULATED WITH TWEEN 80JAMES TREUBIG, FRANCIS BEIDEMAN, ANTHONY GRILLI, SGS LIFE SCIENCE SERVICES, FAIRFIELD, NJ, USA

PO

O

O

O

OO

O

Polysorbate 80w+x+y+z=20

CH(OCH2CH2)yOH

HO(CH2CH2O)w (OCH2CH2)xOH

(OCH2CH2)z

O

O C17H33

FIGURE 1: Volatile Extractables: GC-HS Results

LABEL ANDRETENTION

TIME(MIN)

MAJOR IONS(M/Z)

STRUCTURE LIBRARYMATCH

AMOUNTFOUND(µG/G)

COMMENTS(NAME, EMPIRICAL FORMULA,

NOMINAL MOLECULAR WEIGHT, CAS NUMBER, ETC)

PEAK 316.4

190, 175,

147, 115, 91, 77, 65, 57,

41

913 0.2

1,3-bis(1,1-dimethylethyl)-ben-zene, 1,3-Di-tert-butylbenzene, C14H22, MW 190, CAS 1014-60-4, degradation from antioxi-

dant

PEAK 421.1

206, 191, 163,

115, 91, 77, 57,

41

911 0.1

2,4-bis(1,1-dimethylethyl)-phe-nol, 2,4-Di-tert-butylphenol,

C14H22O, MW 206, CAS 96-76-4, antioxidant

LABEL ANDRETENTION

TIME(MIN)

MAJOR IONS(M/Z)

STRUCTURE LIBRARYMATCH

AMOUNTFOUND(µG/G)

COMMENTS(NAME, EMPIRICAL FORMULA,

NOMINAL MOLECULAR WEIGHT, CAS NUMBER, ETC)

PEAK 316.8

206, 191, 163, 115, 91, 74, 57,

41

970 0.4

2,4-bis(1,1-dimethylethyl)-phe-nol, 2,4-Di-tert-butylphenol,

C14H22O, MW 206, CAS 96-76-4, antioxidant

PEAK 420.6

429, 342, 281, 261, 232, 217, 205, 189, 175, 133, 91, 57, 41

836 2.0

7,9-Di-tert-butyl-1-oxaspiro[4,5]deca-6,9-diene-2,8-dione, C17H24O3, MW 276, CAS

82304-66-3, tentative identifica-tion, low library match, degrada-

tion from antioxidant

FIGURE 2: Semi-Volatile and Non-Volatile Extractables: GC-MS Results of Ethanol Extraction

GC-MS: Gas chromatography-mass spectrometry.P

O

O

O

O

OO

O

Polysorbate 80w+x+y+z=20

CH(OCH2CH2)yOH

HO(CH2CH2O)w (OCH2CH2)xOH

(OCH2CH2)z

O

O C17H33

LABEL ANDRETENTION

TIME(MIN)

MAJOR IONS(M/Z)

STRUC-TURE

LIBRARYMATCH

AMOUNTFOUND(µG/G)

COMMENTS(NAME, EMPIRICAL FORMULA,

NOMINAL MOLECULAR WEIGHT, CAS NUMBER, ETC)

PEAK 313.5

190, 175, 147, 115,

91, 77, 65, 57, 41

945 75.7

2,4-bis(1,1-dimethylethyl)- phenol, 2,4-Di-tert-butylphenol, C14H22O, MW 206, CAS 96-76-

4, antioxidant

PEAK 416.8

206, 191, 163, 115, 91, 74, 57

948 65.6

7,9-Di-tert-butyl-1-oxaspiro[4,5]deca-6,9-diene-2,8-dione, C17H24O3, MW 276, CAS

82304-66-3, tentative identifica-tion, low library match, degrada-

tion from antioxidant

PEAK X33.4

663, 648, 592, 535, 367, 316, 253, 191,

147, 91, 57

Not matched

in the library

Not ob-served by

GC-FID

Oxidized Irgafos 168, C42H63O4P, MW 662, charac-teristic doubly charged ion ob-

served at m/z 316. (Note: Irgafos 168 (structure shown) is Tris(2,4-

ditert-butylphenyl) phosphite, C42H63O3P, MW 646, CAS

31570-04-4, typical antioxidant)

FIGURE 3: Semi-Volatile and Non-Volatile Extractables: GC-MS Results of Ethanol Extraction

GC-MS: Gas chromatography-mass spectrometry; GC-FID: Gas chromatography-flame ionizing detection.

PO

O

O

O

OO

O

Polysorbate 80w+x+y+z=20

CH(OCH2CH2)yOH

HO(CH2CH2O)w (OCH2CH2)xOH

(OCH2CH2)z

O

O C17H33

FIGURE 4 : High-pressure liquid chromatography with ultra-violet (HPLC-UV)

• Reverse phase gradient separates the components of interest, but the limit of detection (LOD = ~2 ppm) was higher than the targeted concentration of 0.15 ppm.

• Solid-phase extraction (SPE) did not sufficiently concentrate the ana-lyte because Tween, being a surfactant, competes with the analytes and saturates the active sites on the SPE cartridge

FIGURE 5: Gas chromatography-flame ionization detection (GC-FID)

• Liquid-liquid extraction to concentrate the analytes was not possible at 150 ppb because Tween shares the solubility characteristics of the ex-tractables targeted.

• Similarly, solid phase extraction to concentrate the analyte because Tween, being a surfactant, competes with the analytes and saturates the active sites on the SPE cartridge.

• These solvent studies demonstrated:• Ideal extraction recovery (100%) was achieved using toluene as the solvent without Tween. • No more than 10-20% recovery was achieved with the target at 0.15 ppm.

pA

MIN